The long term objective of this proposal is to clarify the cellular and molecular mechanisms by which bone marrow failure occurs in patients with Fanconi Anemia (FA). These studies will focus on defining the functional competencies of both general compartments of hematopoiesis; progenitor cells (Aim I) and hematopoietic stromal cells (Aim 2). Aim 1. Preliminary studies in our laboratory have demonstrated that antisense FACC oligonucleotides inhibit clonal growth of committed hematopoietic progenitor cells in vitro but do not inhibit growth of fibroblasts, mitogen stimulated lymphocytes, or endothelial cells. We therefore hypothesize that products of the normal homologs of the FA genes play direct roles in governing growth and differentiation of hematopoietic stem cells and progenitors. This hypothesis will be tested first using both loss-of-function analysis and subsequently by gain-of- function studies using complementation group C progenitor cells. loss-of- function analyses will be carried out first in an attempt to determine whether (a) multipotential (long-term-culture initiating cells) are also sensitive to the antisense molecule, (b) the FACC gene product supports survival of cells in a growth factor depleted environment, and to locate the specific downstream block point imposed by the antisense molecules. After confirming that the block points identified using antisense oligonucleotides are recapitulated in progenitor cells from peripheral blood, cord blood, or bone marrow cells from FA patients, we will perform gain-of-function analyses using PSFFVFACC, an amphotropic retrovirus constructed in our laboratory designed to enforce expression of normal FACC in FA cells of the complementation group. Aim 2. Preliminary studies in our laboratory have identified stimulus (IL- l)-response (hematopoietic growth factors) blunting in some fibroblasts from FA patients. Because the defects were not consistent between affected siblings in multiplex families, we reason that the defects were acquired. Consequently, we hypothesize that (l) bone marrow stromal cells from some patients with FA fall to produce an adequate quantity of normal hematopoietic growth factors and (2) this stromal cell defect results from somatic cell injury, mutation or dysfunction and not from a related (co-factor) function of the FA gene products themselves. Our second aim is designed to test these hypotheses using stromal fibroblasts and macrophages from the marrow and blood, respectively, from FA patients. Using normal fibroblasts and monocytes in loss-of-function studies we will test the notion that the defects observed do not derive from any particular specific function of the FA proteins.